The divalent cation, Ca2 ions, has long been recognized as a primary intracellular signal through which stimuli evoke cellular responses such as endo- and exocytosis, motility, alterations in intermediary metabolism, and proliferation. However, the biochemial mechanisms through which stimulus-induced fluxes in free Ca 2 ion concentration are coupled to cellular regulation have only recently been elucidated. Work in this and other laboratories has revealed calmodulin (CaM) to be a major intracellular receptor in eukaryotes for these Ca2 ion-regulatory signals. Studies in this and other laboratories summarized in Significance and Progress Report, have provided substantial information concerning the general mechanism through which CaM acts. However, limited information is available concerning the exact molecular structures which provide for CaM-enzyme interaction or how this interaction induces enzyme activation. Such information is essential to understand Ca-dependent regulation and to elucidate potential dysfunctions in this regulation which are causative factors in disease processes. The major goal of the research plan presented in Methods is to reveal the structural interactions of CaM and CaM regulated enzymes which result in Ca.CaM-dependent stimulation of enzyme activity. Structural, biochemical and immunological studies, designed to yield a further understanding of these processes, are proposed including: 1) Detailed comparative structural analyses of protozoan, plant and if isolated, prokaryotic CaMs and similar but distinct Ca-binding proteins found in C. elegans and vertebrate smooth muscles. 2) Studies of the exact role of CaM in regulating ciliary dynein ATPase activity. 3) Detailed analyses, using immobilized phenothiazines, of the mechanism of action of neuroleptic drugs both on CaM and other proteins which may be shown to bind phenothiazines specifically. 4) Detailed chemical, enzymological and immunological studies of the CaM binding domains in a number of CaM-regulated enzymes.
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